This invention relates to new compounds, which are useful in the treatment of cancer. These compounds are used to increase the effect of conventional cytotoxic pharmaceuticals.
Cytostatic or cytotoxic compounds are widely used in the treatment of cancer. Doxorubicin is an aminoglycosidic anthracycline antibiotic and will be used as a typical representative of this group of compounds.
The cell membrane represents a physical barrier and there are some factors that determine the rate of uptake of doxorubicin. The main factors are hydrophobicity (an increase will increase the rate of uptake) and protonation degree of amino groupxe2x80x94pKa (a decrease will increase the rate of entry). The doxorubicin inhibits cell growth and has a marked effect on the nuclear material, which becomes non-specifically thickened, agglutinated or broken. The major binding force between doxorubicin and DNA is intercalation of the planar chromophore, stabilised by an external electrostatic binding of the positive charged amino sugar residue with negative phosphate group of DNA.
The intercalated drug molecules appear to prevent the changes in conformation of the helix, which are necessary as a preliminary to initiation of nucleic acid synthesis. The major lethal effect of doxorubicin is inhibition of nucleic acid synthesis. As consequence the drug is more active against dividing cells and the greatest effect is in the S stage of the cell cycle (Brown J. R., Adriamycin and related anthracycline antibiotics in: Progress in Medicinal Chemistry edited by G. P. Ellis and G. B. West, Elsevier/North-Holland Biomedical Press v. 15, pp.125-164, 1978).
Some observations are consistent with the formation complex of electrostatic nature between the positive amino group of doxorubicin and negative phosphate group of phospholipids such as cardiolipin, phosphatidyl serine, phosphatidyl inositol and phosphatidic acid. Cardiolipin is an almost characteristic component of the inner membrane of mitochondria, which are abundant in the cardiac muscle. The pathogenesis of the mitochondrial lesions is one of the major and more specific sub-cellular changes characterizing doxorubicin cardiotoxicity. The rather selective toxicity doxorubicin for mitochondria may be due to the high concentrations of cardiolipin in the mitochondria of the cardiac muscle (Duarte-Karim M., et al. Biochem. Biophys. Res. Comm., v.71, N.2, pp.658-663, 1976).
The interaction between doxorubicin and lipids has been studied using large unilamellar vesicles (LUVET) composed of mixtures of anionic phospholipids and various zwitterionic phospholipids. Dilution of anionic lipids with zwitterionic lipids leads to decreased membrane association of the drug because electrostatic forces are very important in doxorubicin-membrane interaction. However, binding of doxorubicin to LUVET composed of anionic phospholipids combined with phosphatidylethanolamine (PE) is much higher than binding to LUVET made of anionic lipids plus a range of other zwitterionic lipids such as phosphatidylcholine and the N-methyethanolamine and N,N-dimethylethanolamine derivatives of PE (Speelmans G, et al., Biochemistry, v.36, N.28, pp.8657-8662, 1997).
The interaction of adriamycin with human erythrocytes was investigated in order to determine the membrane binding sites and the resultant structural perturbation. Electron microscopy revealed that red blood cells incubated with the therapeutic concentration of the drug in human plasma changed their discoid shape to both stomatocytes and echinocytes. The drug was incubated with molecular models. One of them consisted of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine multilayers, representatives of phospholipid classes located in the outer and inner leaflets of the erythrocyte membrane, respectively. X-ray diffraction showed that adriamycin interaction perturbed the polar head and acyl chain regions of both lipids. It is concluded that adriamycin incorporates into both erythrocyte leaflets affecting its membrane structure (Suwalskly M., Z Naturforsch [C] v.54, N3-4, pp.271-277, 1999).
The different physicochemical properties of dipalmitoylphosphatidylcholine liposomes with soybean-derived sterols have been studied. Liposomal doxorubicin increased the pharmacological effect compared with free drug, suggesting a decrease of side effect and long circulation (Maitani Y., Yakugaku Zasshi, v. 116, N.12, pp.901-910, 1996).
Liposomes containing polyethylene glycol-derivatised phospholipids are able to evade the reticulo-endothelial system and thereby remain in circulation for prolonged periods. The doxorubicin encapsulated in these sterically stabilised liposomes suppresses the growth of established human lung tumour xenografts in severe combined immunodeficient mice and inhibits the spontaneous metastases of these tumours (Sakakibara T., et al., Cancer Res., v.56, N.16, pp.3743-3746, 1996).
A liposome encapsulation can protect surrounding tissue from the cytotoxic effects of the drugs after subcutaneous (s.c.) administration. Liposomes composed of xe2x80x9cfluid-statexe2x80x9d phospholipids only delayed the damaging effects of doxorubicin when injected s.c. Liposomes with a more rigid nature were much more effective in preventing local tissue damage over a longer period of time when administered s.c. (Oussoren C., et al., Biochim. Biophys. Acta, v.1369, N.1, pp.159-172, 1998).
Exogenous polyunsaturated fatty acids modulate the cytotoxic activity of anti-cancer drugs in the human breast cancer cell line MDA-MB-231. Among all polyunsaturated fatty acids tested, docosahexaenoic acid was the most potent in increasing doxorubicin cytotoxicity (E. Germain, et al., Int. J. Cancer, v.75, pp. 578-583, 1998).
There remains a need for novel compounds and methods for the treatment of cancer. The present invention aims i.a. to increase the pharmacological activity of presently used anti-cancer drugs, such as doxorubicin, and to introduce novel approaches to the treatment of cancer.
The present invention makes available new compounds and new combinations of compounds, which, together with known cytotoxic or cytostatic pharmaceuticals, introduce improved possibilities to combat cancer. Further, the present invention discloses a method of synthesis of these compounds, and a modified form of a cytostatic pharmaceutical compound; doxorubicin.
It has been shown that the amount of lipoperoxides arise after the action of doxorubicin (DXR) in the presence of docosahexaenoic acid and oxidants, in the human breast cancer cells (line MDA-MB-231). This may endow tumour cells with metabolic characteristics that decrease their propensity to survive the effects of doxorubicin.
The present inventor has previously made available novel amides of the all-trans-retinoic acid or 13-cis-retinoic acid, arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid or linolenic acid with 2-aminoehtanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine containing phosphate groups (SE 9900941-7, filed on Mar. 16, 1999). The present invention discloses the use of specific compounds, in particular their application for increase the pharmacological activity of doxorubicin.
These novel compounds contain hydrophobic residues of polyunsaturated fatty acids, retinoic acid residues and a phosphate group, which has a negative charge. Thus, the interaction between molecules of novel compounds and doxorubicin could be realised by hydrophobic interaction between fatty acid residues or retinoic acid residues and the planar chromophore of doxorubicin, as well as an electrostatic interaction between contrary charged functional groups both compounds.
On the one hand these binary complexes have all necessaries properties for directed transport through the membrane of the cancer cells and resemble a xe2x80x9cTrojan horsexe2x80x9d. On the other hand, the dissociation of these binary complexes inside of the cancer cells releases xe2x80x9cnativexe2x80x9d, positive charged molecules of doxorubicin, with the result that favourable conditions for doxorubicin intercalation into DNA are created.
The following compounds 1 through 4, 1a through 4a, and 5 through 20 form the basis of the invention. They havexe2x80x94in partxe2x80x94been disclosed in the Swedish patent application no. 9900941-7, filed on Mar. 16, 1999.
Retinoic acid derivatives:
1. N-(all-trans-retinoyl)-o-phospho-2-aminoethanol
1a. N-(13-cis-retinoyl)-o-phospho-2-aminoethanol
2. N-(all-trans-retinoyl)-o-phospho-L-serine
2a. N-(13-cis-retinoyl)-o-phospho-L-serine
3. N-(all-trans-retinoyl)-o-phospho-L-threonine
3a. N-(13-cis-retinoyl)-o-phospho-L-threonine
4. N-(all-trans-retinoyl)-o-phospho-L-tyrosine
4a. N-(13-cis-retinoyl)-o-phospho-L-tyrosine
Arachidonic acid derivatives:
5. N-arachidonoyl-o-phospho-2-aminoethanol
6. N-arachidonoyl-o-phospho-L-serine
7. N-arachidonoyl-o-phospho-L-threonine
8. N-arachidonoyl-o-phospho-L-tyrosine
Docosahexaenoic acid derivatives:
9. N-docosahexaenoyl-o-phospho-2-aminoethanol
10. N-docosahexaenoyl-o-phospho-L-serine
11. N-docosahexaenoyl-o-phospho-L-threonine
12. N-docosahexaenoyl-o-phospho-L-tyrosine
Eicosapentaenoic acid derivatives:
13. N-eicosapentaenoyl-o-phospho-2-aminoethanol
14. N-eicosapentaenoyl-o-phospho-L-serine
15. N-eicosapentaenoyl-o-phospho-L-threonine
16. N-eicosapentaenoyl-o-phospho-L-tyrosine
Linoleic acid derivatives:
17. N-linolenoyl-o-phospho-2-aminoethanol
18. N-linolenoyl-o-phospho-L-serine
19. N-linolenoyl-o-phospho-L-threonine
20. N-linolenoyl-o-phospho-L-tyrosine
In the following description and examples, the above compounds are referred to as C1 through C4, C1a-C4a, and C5-C20.
A study of the anti-tumour effect of complexes between doxorubicin (DXR) and any one of the above compounds C1-C4, C1a-C4a, and C5-C20, as compared with DXR alone, was carried out using mice with EAC (Ehrlich ascites carcinoma). The extent of inhibition of EAC growth in mice, achieved by the tested compounds, compared to DXR, was used for evaluation of the anti-tumour activity of each tested DXR/compound complex.
It has been experimentally shown that the complex DXR with any compound alone (C1-C4, C1a-C4a, and C5-C20) did not display an anti-tumour effect. In particular, in the DXR/C4 complex, the compound C4 cancelled the anti-tumour action of DXR and even exhibited some (insignificantly small) stimulating influence on EAC growth in mice. In the DXR/C5 complex, compound C5 cancelled the anti-tumour action of DXR.
The present inventor has however shown that complexes of DXR and C4 or C4a, together with C5; DXR/C4 (C4a) with C9; DXR/C4 (C4a) with C13; and DXR/C4 (C4a) with C17 display anti-tumour effects.
The attached series of experimental results, support this finding. The following doses were used:
DXRxe2x80x943.5 mg/kg of body weight
C4xe2x80x948.15 mg/kg of body weight
At the molar ratios C4:C5 equal to 1:3; 1:2.9; 1:2.8; 1:2.7 and 1:2.6, the EAC growth inhibition was 45.0%; 43.6%; 46.0%; 48.2%; 50.4%, respectively.
At the molar ratios C4:C5: equal to 1:2.5; 1:2.4; 1:2.3 and 1:2.2, the EAC growth inhibition was 48.7%; 51.5%; 53.4% and 57.0%, respectively.
At the molar ratios C4:C5: equal to 1:2.1; 1:2; 1:1.9 and 1:1.8, the EAC growth inhibition was 58.0%; 59.5%; 62.0% and 65.2%, respectively.
At the molar ratios C4:C5 equal to 1:1.7; 1:1.6; 1:1.5 and 1:1.4, the EAC growth inhibition was 67.1%; 67.9%; 69.1% and 70.0%, respectively.
At the molar ratios C4:C5 equal to 1:13; 1:1.2; 1:1.1 and 1:1, the EAC growth inhibition was 69.4%; 66.7%; 62.5% and 65.2%, respectively.
It should be noted that, at the molar ratios C4:C5 equal to 1:1.7; 1:1.6; 1:1.5; 1:1.4 and 1:1.3, the EAC growth inhibition with reference to the DXR group (positive control) was 38.8%; 40.3%; 42.6%; 44.2% and 41.1%, respectively.
DXRxe2x80x943.5 mg/kg of body weight
C5xe2x80x946.4 mg/kg of body weight
At the molar ratios C4:C5 equal to 1:1; 1.1:1; 1.2:1; 1.3:1 and 1.4:1, the EAC growth inhibition was 65.2%; 61.6%; 66.2%; 69.3% and 69.8%, respectively.
At the molar ratios C4:C5 equal to 1.5:1; 1.6:1; 1.7:1 and 1.8:1, the EAC growth inhibition was 74.9%; 78.1%; 74.1% and 66.4%, respectively.
At the molar ratios C4:C5 equal to 1.9:1; 2:1; 2.1:1 and 2.2:1, the EAC growth inhibition was 65.1%; 61.8%; 63.2% and 58.0%, respectively.
At the molar ratios C4:C5 equal to 2.3:1; 2.4:1; 2.5:1 and 2.6:1, the EAC growth inhibition was 55.5%; 58.7%; 56.7% and 57.6%, respectively.
At the molar ratios C4:C5 equal to 2.7:1; 2.8:1; 2.9:1 and 3:1, the EAC growth inhibition was 56.6%; 55.0%; 50.4% and 45.3%, respectively.
It should be noted that, at the molar ratios C4:C5 equal to 1.3:1; 1.4:1; 1.5:1; 1.6:1 and 1.7:1, the EAC growth inhibition with reference to the DXR group (positive control) was 43.2%; 44.1%; 51.4%; 57.7% and 49.8%, respectively.
In particular, some of the experiments for testing the anti-tumour effects of DXR/C4+C9 complexes, DXR/C4+C13 complexes, DXR/C4+C17 complexes, DXR/C4a+C5 complexes indicate this.
DXR/C4+C9 complexes:
DXRxe2x80x943.5 mg/kg of body weight, and C4xe2x80x948.15 mg/kg of body weight
At the molar ratios C4:C9 equal to 1:1; 1:1.4; 1:1.8 and 1:2.3, the EAC growth inhibition was 61.8%; 67.3%; 62.5% and 49.7%, respectively.
At the molar ratio C4:C9 equal to 1:1.4, the EAC growth inhibition with reference to the DXR group (positive control) was 49.3%.
DXR/C4+C13 complexes:
DXRxe2x80x943.5 mg/kg of body weight, and C4xe2x80x948.15 mg/kg of body weight.
At the molar ratios C4:C13 equal to 1:1.3; 1:1.6; 1:2 and 1:2.5, the EAC growth inhibition was 67.0%; 64.6%; 54.2% and 45.1%, respectively.
At the molar ratios C4:C13 equal to 1:1.3 and 1:1.6, the EAC growth inhibition with reference to the DXR group (positive control) was 47.6% and 43.8%, respectively.
DXR/C4+C17 complexes:
DXRxe2x80x943.5 mg/kg of body weight, and C17xe2x80x946,0 mg/kg of body weight.
At the molar ratios C4:C17 equal to 1:1; 1.4:1; 2:1 and 2.7:1, the EAC growth inhibition was 62.9%; 67.4%; 60.3% and 51.8%, respectively.
At the molar ratio C4:C17 equal to 1.4:1, the EAC growth inhibition with reference to the DXR group (positive control) was 45.0%.
DXR/C4a+C5 complexes:
DXRxe2x80x943.5 mg/kg of body weight, and C5xe2x80x946,4 mg/kg of body weight.
At the molar ratios C4a:C5 equal to 1.2:1; 1.6:1; 1.9:1 and 2.5:1, the EAC growth inhibition was 64.4%; 72.4%; 62.2% and 52.5%, respectively.
At the molar ratio C4a:C5 equal to 1.6:1, the EAC growth inhibition with reference to the DXR group (positive control) was 49.4%.
The present inventor has shown that DXR/(C4+C4a)+C5 complexes; DXR/(C4+C4a)+(C5+C9+C13) complexes; DXR/(C4+C4a)+(C5+C9+C13+C17) complexes display an anti-tumour effect. The experiments for testing the anti-tumour effect support this finding.
DXR/(C4+C4a)+C5 complexes: DXRxe2x80x943.5 mg/kg of body weight
C5xe2x80x946.4 mg/kg of body weight
At the molar ratios (C4+C4a):C5 equal to 1.2:1; 1.6:1; 1.9:1 and 2.5:1 EAC growth inhibition was 63.1%; 71.6%; 61.0% and 47.3%, respectively.
At the molar ratios (C4+C4a):C5 equal to 1.6:1 EAC growth inhibition with reference to DXR group (positive control) was 53.1%.
DXR/(C4+C4a)+(C5+C9+C13) complexes: DXRxe2x80x943.5 mg/kg of body weight
(C4+C4a)xe2x80x948.15 mg/kg of body weight
At the molar ratios (C4+C4a):(C5+C9+C13) equal to 1:1; 1:1.4; 1:1.8 and 1:2.3 EAC growth inhibition was 60.1%; 68.3%; 61.3% and 47.2%, respectively.
At the molar ratios (C4+C4a):(C5+C9+C13) equal to 1:1.4 EAC growth inhibition with reference to DXR group (positive control) was 49.8%.
DXR/(C4+C4a)+(C5+C9+C13+C17) complexes: DXRxe2x80x943.5 mg/kg of body weight
(C4+C4a)xe2x80x948.15 mg/kg of body weight
At the molar ratios (C4+C4a):(C5+C9+C13+C17) equal to 1:1.3; 1:1.6; 1:2 and 1:2.5 EAC growth inhibition was 68.0%; 69.7%; 56.3% and 43.5%, respectively.
At the molar ratios (C4+C4a):(C5+C9+C13+C17) equal to 1:1.3 and 1:1.6 EAC growth inhibition with reference to DXR group (positive control) was 45.3% and 48.1% respectively.
Investigations of the anti-tumour effects of DXR/C4+C5 complexes, DXR/C4+C9 complexes, DXR/C4+C13 complexes, DXR/C4+C17 complexes, DXR/C4a+C5 complexes allow the following conclusion:
At the molar ratios C4(or C4a): C5 (or C9 or C13 or C17) from 1:3 to 1:1 and from 1:1 to 3:1, the anti-tumour action complexes exceed the effect of DXR alone. The results show significant effects for complexes within the intervals 1:2-1:1 and 1:1-2:1, with improved effects corresponding to the more narrow intervals 1:1.7-1:1.3 and 1.3:1-1.7:1.
The investigation of anti-tumour effects of DXR/(C4+C4a)+C5 complexes, DXR/(C4+C4a)+(C5+C9+C13) complexes, DXR/(C4+C4a)+(C5+C9+C13+C17)complexes confirm this conclusion. Moreover, it is true for complexes, in which from two to six compounds (4, 4a, 5, 9, 13, 17) participate in interactions with DXR.
These results-were obtained at such concentrations of novel compounds (C4, C4a, C5, C9, C13, C17) which are considerably above their critical micelle concentrations.
A water-soluble aromatic compound, such as doxorubicin could be in the form of micelle in water solutions. The present inventor has surprisingly found, that doxorubicin in a concentration interval of 1-2 mg/ml forms mixed micelles with the novel compounds (C4, C4a, C5, C9, C13, C17) more effectively. Further, the anti-tumour activity of these mixed micelles is significantly higher then for doxorubicin alone in the same concentrations.
Thus, mixed micelles consisting of an amide of the all-trans-retinoic acid or/and an amide of the 13-cis-retinoic acid with O-phospho-L-tyrosine (C4 or C4a; C4+C4a), amides of polyunsaturated acids with O-phosphorylethanolamine (C5 or C9 or C13 or C17; C5+C9; C5+C13; C5+C17; C9+C13; C9+C17; C13+C17; C5+C9+C13; C5+C9+C17; C5+C13+C17; C9+C13+C17; C5+C9+C13+C17) and doxorubicin, display anti-tumour activity.
Thus, mixed micelles consisting of amide of the all-trans-retinoic acid or 13-cis-retinoic acid with O-phospho-L-tyrosine (C4 or C4a), amide polyunsaturated acid with O-phosphorylethanolamine (C5 or C9 or C13 or C17) and doxorubicin, display anti-tumour activity.
The inventor has previously described a universal method of synthesis amides of retinoic acids and polyunsaturated acids with hydroxyamino acids and ethanolamine containing phosphate groups. In the present application, the inventor discloses a method of synthesis of the N-acyl-O-phospho-2-aminoethanol and the N-retinoyl-O-phospho-L-tyrosine.
That method has the following advantages in comparison with previous one
the synthesis is performed in one step
the yield is high, reaching 90%,
simplified and time-saving synthesis
the final product can be purified without chromatography